The proposed research will provide new and comprehensive insight into debranching enzyme (Dbr1) that is a metalloenzyme central to the processing and biogenesis of regulatory RNAs. The use of modified branched RNA (bRNA) substrates and mimics will allow biochemical, structural and single-molecule studies into the binding, recognition and catalytic cleavage mechanism of Dbr1. This dissection of the molecular underpinnings of Dbr structure, function and dynamics is critical to the understanding of the unique specificity of Dbr1 and its active-site in cleavage of the bRNA 2'-5'-phophodiester cleaved. Through unprecedented access to synthetic bRNA, biochemical studies are proposed here to evaluate the active site residues and substrate requirements of Dbr1. Crystallographic analysis of Dbr1 will inform and enhance these biochemical studies by providing molecular details of protein and RNA contacts relevant to recognition, specificity and cleavage. Further, single-molecule spectroscopy will probe the dynamics of the macromolecular interactions to furnish important details into the reaction pathway and mechanism. The trifecta of methods proposed will provide a synergistic and cohesive account of the molecular details of Dbr1 cleavage of bRNA. These molecular details are critical for interventions through designed inhibitors in future therapeutic paradigms.